U.S. patent application number 16/375107 was filed with the patent office on 2019-07-25 for compositions and methods relating to synthetic rna polynucleotides created from synthetic dna oligonucleotides.
This patent application is currently assigned to New England Biolabs, Inc.. The applicant listed for this patent is New England Biolabs, Inc.. Invention is credited to Isaac B. Meek, G. B. Robb, Ezra Schildkraut, Dianne S. Schwarz.
Application Number | 20190225961 16/375107 |
Document ID | / |
Family ID | 59960254 |
Filed Date | 2019-07-25 |
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United States Patent
Application |
20190225961 |
Kind Code |
A1 |
Robb; G. B. ; et
al. |
July 25, 2019 |
Compositions and Methods Relating to Synthetic RNA Polynucleotides
Created From Synthetic DNA Oligonucleotides
Abstract
Compositions and methods are provided for forming a single RNA
polynucleotide from a plurality of DNA oligonucleotides in a single
reaction chamber using combined reagents in a single step reaction.
DNA polymerase, RNA polymerase and single stranded (ss) DNA
oligonucleotides are combined where each DNA oligonucleotide has
one or more sequence modules, wherein one sequence module in the
first ss DNA oligonucleotide is complementary to a sequence module
at the 3' end of the second ss DNA oligonucleotide; and wherein a
second module on the first ss DNA oligonucleotide is an RNA
polymerase promoter sequence; and forming a single RNA
polynucleotide, excluding the RNA promoter sequence, derived from
the first and second DNA oligonucleotides
Inventors: |
Robb; G. B.; (Somerville,
MA) ; Meek; Isaac B.; (Hopkinton, MA) ;
Schwarz; Dianne S.; (Watertown, MA) ; Schildkraut;
Ezra; (Cerillos, NM) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
New England Biolabs, Inc. |
Ipswich |
MA |
US |
|
|
Assignee: |
New England Biolabs, Inc.
Ipswich
MA
|
Family ID: |
59960254 |
Appl. No.: |
16/375107 |
Filed: |
April 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15469681 |
Mar 27, 2017 |
10301619 |
|
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16375107 |
|
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62317035 |
Apr 1, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/13 20130101;
C12Q 1/68 20130101; C12N 2310/20 20170501; C12N 15/1068 20130101;
C12N 9/96 20130101; C12N 2310/16 20130101; C12N 2330/31 20130101;
C12P 19/34 20130101; C12N 9/22 20130101; C12N 2330/00 20130101;
C12N 15/115 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12N 9/22 20060101 C12N009/22; C12N 9/96 20060101
C12N009/96; C12N 15/115 20060101 C12N015/115; C12P 19/34 20060101
C12P019/34 |
Claims
1. A method of forming a single RNA polynucleotide from a plurality
of DNA oligonucleotides in a single reaction chamber in a single
step reaction, comprising: (a) combining with a DNA polymerase and
an RNA polymerase, a first and second synthetic single strand (ss)
DNA oligonucleotide, each having one or more sequence modules,
wherein one sequence module in the first ss DNA oligonucleotide is
complementary to a sequence module at the 3' end of the second ss
DNA oligonucleotide; and wherein a second module on the first ss
DNA oligonucleotide is an RNA polymerase promoter sequence; and (b)
forming a single RNA polynucleotide derived from the first and
second DNA oligonucleotides excluding the RNA promoter.
2. A method according to claim 1, wherein the single RNA
polynucleotide is a guide RNA.
3. A method according to claim 1, further comprising a Cas nuclease
capable of being activated in the presence of the guide RNA.
4. A method according to claim 1, further comprising adding the Cas
nuclease in step (a)
5. A method according to claim 1, wherein the single RNA
polynucleotide is selected from the group consisting of a guide
RNA, an aptamer, a mRNA, a tRNA, a microRNAs a shRNA, an snRNA, a
short non-coding RNA, a long non-coding RNA, an RNA probe, and a
ribozyme.
6. A method according to claim 1, further comprising combining a
third synthetic ss DNA oligonucleotide and a fourth synthetic ss
DNA oligonucleotide in the reaction chamber, wherein the 5' end of
the third synthetic ss DNA oligonucleotide hybridizes to the 5' end
of the second ss DNA oligonucleotide and the 3' end of the third ss
DNA oligonucleotide hybridizes to the 3' end of the fourth ss DNA
oligonucleotide for forming a single RNA polynucleotide comprising
the sequences from first, second, third and fourth oligonucleotide
excluding the RNA promoter sequence.
7. A method according to claim 1, wherein the DNA polymerase is a
strand displacing polymerase.
8. A method according to claim 6, further comprising a DNA ligase
in the reaction chamber.
9. A method according to claim 1, further comprising (c) combining
a reactor sequence module in the single RNA molecule with a
detector molecule; and causing a detectable signal.
10. A method according to claim 9, wherein the detector molecule is
a third sequence module on the first, second, third or fourth ss
DNA oligonucleotide.
11. A method according to claim 10, wherein the reactor sequence
module is a first aptamer.
12. A method according to claim 9, wherein the detector molecule is
a second aptamer or a fluorescent marker for binding the first
aptamer.
13. A method according to claim 1, further comprising immobilizing
the 5' end of the RNA polynucleotide on a solid support.
14. A method according to claim 1, wherein the 5' end of the RNA
polynucleotide comprises a sequence module containing a modified
nucleotide.
15. A method according to claim 14, wherein the modified nucleotide
is biotin or desthiobiotin.
16. A method according to claim 1, wherein the first ss DNA
oligonucleotide contains a sequence module in which the sequence is
variable.
17. A method according to claim 1, wherein the variable sequence
module is DNA targeting sequence for a Cas nuclease.
18. A method according to claim 1, wherein the first synthetic ss
DNA oligonucleotide has a variable sequence module and the reaction
chamber is one position in an array of reaction chambers so that
whereas each reaction chamber contains a first ss DNA
oligonucleotide, the variable sequence module differs in
substantially each reaction chamber.
19. A method according to claim 18, wherein the second ss DNA
oligonucleotide is optionally the same for each well.
20. A method according to claim 17, wherein the second ss DNA
oligonucleotide comprises a tracrRNA sequence module.
21. A method according to claim 1, further comprising forming a
plurality of different guide RNAs.
22. A method for making an RNA guided protein, comprising: (a)
selecting a protein for guiding to a nucleic acid target using a
guide RNA; (b) making an RNA according to claim 1; (c) associating
the protein with the RNA from (b) to make an RNA guided
protein.
23. A method according to claim 22, wherein the RNA guided protein
is an RNA guided nuclease.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/469,681, filed Mar. 27, 2017, which claims
the benefit of U.S. provisional application Ser. No. 62/317,035
filed Apr. 1, 2016, incorporated by reference herein.
BACKGROUND
[0002] While amplification of target genomic DNA or cDNA in a
library to generate adapter linked DNA that can be copied by RNA
polymerases is routinely performed, preparation of synthetic RNA
polynucleotides de novo involves a series of reactions which can
involve multiple steps and is quite cumbersome. Recently with
increasing need to manufacture and study larger RNA polynucleotides
including guide RNAs and mRNAs, it is desirable to improve the
efficiency of the synthetic methodologies.
SUMMARY
[0003] In general in one aspect, a preparation is provided in a
single reaction vessel, that includes: (a) a first and a second
synthetic single strand (ss) DNA oligonucleotide each containing a
plurality of sequence modules, wherein the first synthetic
oligonucleotide comprises a 5' end and a 3' end, and wherein one
sequence module is positioned at the 3' end of the first
oligonucleotide that hybridizes to a complementary sequence module
at the 3' end of the second oligonucleotide; and a second sequence
module on the first oligonucleotide corresponds to an RNA promoter
sequence; (b) a DNA polymerase capable of extending the 3' end of
the first oligonucleotide and the 3' end of the second
oligonucleotide in a 5' direction to produce a double stranded (ds)
DNA; and (c) an RNA polymerase (RNAP).
[0004] In one aspect, a Cas nuclease is included in the
preparation. In another aspect, the first oligonucleotide contains
a sequence module in which the sequence is variable or the first or
second oligonucleotide contains a sequence module in which the
sequence is variable. The variable sequence may correspond to a
sequence that is complementary to a DNA targeting sequence on a
guide RNA.
[0005] In another aspect, the first or second oligonucleotide
contains a sequence module corresponding to a sequence that is
complementary to tracrRNA.
[0006] In another aspect, the hybridizing sequence of the first
oligonucleotide is less than 15 nucleotides and/or the
non-hybridizing sequence of the first oligonucleotide is at least
15 nucleotides. In another aspect, the second oligonucleotide
sequence has a non-hybridizing sequence, wherein the
non-hybridizing sequence of the second oligonucleotide is at least
15 nucleotides. In another aspect, a third synthetic
oligonucleotide is provided having a predetermined sequence and a
fourth synthetic oligonucleotide with a predetermined sequence,
wherein the 5' end of the third synthetic oligonucleotide
hybridizes to the 5' end of the second oligonucleotide and the 3'
end of the third oligonucleotide hybridizes to the 3' end of the
fourth oligonucleotide.
[0007] In another aspect, the DNA polymerase is a strand displacing
polymerase and in another aspect the preparation includes a DNA
ligase where three or more oligonucleotides are assembled
together.
[0008] In another aspect, at least one of the first, second, third
or fourth oligonucleotide comprise a sequence module that is a
first detector molecule that when transcribed by the RNAP and
combined with a second detector molecule such as a second sequence
module on the first, second, third or fourth oligonucleotide,
causes a detectable signal. An example of a first detector molecule
is an aptamer sequence and of the second detector molecule is a
fluorescent dye. An example of an aptamer sequence is mango or
broccoli. In one aspect, the 5' end of the first oligonucleotide is
immobilized on a solid support. In another aspect, the 5' end of
the oligonucleotide includes a sequence module containing a
modified nucleotide. In another aspect, the modified nucleotide is
biotin or desthiobiotin.
[0009] In general, in one aspect, a method is provided of forming a
single RNA polynucleotide from a plurality of DNA oligonucleotides
in a single reaction chamber in a single step reaction, that
includes: combining at least a first and second synthetic ss DNA
oligonucleotide, each having one or more sequence modules, wherein
one sequence module in the first ss DNA oligonucleotide is
complementary to a sequence module at the 3' end of the second ss
DNA oligonucleotide; and wherein a second module on the first ss
DNA oligonucleotide is an RNAP promoter sequence; and forming a
single RNA polynucleotide derived from the first and second DNA
oligonucleotides excluding the RNAP promoter. In one aspect, the
polymerase is the Klenow fragment of E. coli DNA polymerase I.
[0010] In one aspect, the single RNA polynucleotide is a guide RNA.
In another aspect, a Cas nuclease capable of being activated in the
presence of the guide RNA is included in the reaction chamber or is
added after the single RNA polynucleotide is formed. Examples of
single RNA polynucleotides formed by the method include an RNA
selected from the group consisting of a guide RNA, an aptamer, a
mRNA, a tRNA, a microRNA, a shRNA, an snRNA, a short non-coding
RNA, a long non-coding RNA, an RNA probe, and a ribozyme.
[0011] In one aspect, a third and fourth synthetic ss DNA
oligonucleotide may be included in the reaction chamber. The third
synthetic ss DNA oligonucleotide may have a sequence module at the
5' end that hybridizes to the 5' end of the second ss DNA
oligonucleotide. The 3' end of the third ss DNA oligonucleotide may
include a sequence module that hybridizes to the 3' end of the
fourth ss DNA oligonucleotide. The polymerase may fill-in the
hybridized first, to fourth ss DNA oligonucleotide to form a duplex
DNA which can be transcribed to form a single RNA polynucleotide
that does not include the RNA promoter sequence. The method is not
intended to be limited to four ss DNA oligonucleotides. In addition
to assembling two or four ss DNA oligonucleotides described above,
it is possible to utilize any number of odd or even numbered
oligonucleotides beyond 2 oligonucleotides as desired.
[0012] If the polymerase is capable of strand displacement (such as
Bst polymerase, Bst large fragment or Bst mutants, Deep Vent.RTM.
polymerase, Vent.RTM. polymerase, Klenow fragment of E. coli DNA
polymerase I (all commercially available from New England Biolabs,
Ipswich, Mass.)), an intact duplex will be formed. If the
polymerase is not capable of strand displacement (for example,
Phusion.RTM. (Thermo Fisher Scientific), T7 DNA polymerase, T4 DNA
polymerase, Taq polymerase (all commercially available from New
England Biolabs, Ipswich, Mass.)) it will be desirable to include a
ligase to repair nicks in order to create a full length intact
duplex DNA from which an RNA can be transcribed.
[0013] In another aspect, a sequence module in the single RNA
polynucleotide is a reactor sequence such as an aptamer, such as
mango or broccoli, capable of combining with a detector molecule
such as a fluorescent dye or a second aptamer, to give a detectable
signal. Alternatively, the detector molecule may be another
sequence module in the single RNA polynucleotide.
[0014] In one aspect, the RNA polynucleotide is immobilized on a
solid support. In another aspect, the 5' end of the RNA
polynucleotide contains a modified nucleotide for example, biotin
or desthiobiotin.
[0015] In one aspect, the first ss DNA oligonucleotide contains a
sequence module that contains a variable sequence. The RNA
polynucleotide may be a guide RNA for Cas nuclease and the variable
sequence may include a sequence suitable for targeting a DNA or
RNA.
[0016] In one aspect, a library of first or second ss DNA
oligonucleotides are provided in the method where substantially
each member of the library has a different variable sequence
module. Each reaction chamber assembled in an array contains one of
these members. If the variable sequence module is in the first ss
DNA oligonucleotide, a second ss DNA oligonucleotide, a DNA
polymerase, an RNAP and optionally a Cas nuclease are also added to
each reaction chamber in the array. At least one of the ss DNA
oligonucleotides further includes a sequence module corresponding
to a tracrRNA sequence to allow the RNA polynucleotide to interact
with the Cas nuclease, activating the Cas nuclease to guide it to
its target and enable it to cleave. The RNA polynucleotides in the
library associated with Cas can then be tested to determine which
if any binds a desired sequence in a genome.
[0017] In general in one aspect, a method is provided for making an
RNA chimera from a plurality of synthetic DNA oligonucleotides,
that includes (a) hybridizing overlapping sequences of synthetic
DNA oligonucleotides; wherein (i) the 3' end of a first synthetic
DNA oligonucleotide hybridizes to a 3' end of a second synthetic
oligonucleotide; and the 5' end of the first oligonucleotide
comprises a non-hybridizing sequence, containing an RNA promoter;
(ii) optionally the 3' end of a third oligonucleotide hybridizes to
the 3' end of a fourth oligonucleotide; (b) extending by means of a
polymerase, the 3' ends of each oligonucleotide in a 5' direction
to produce ds DNA; and (c) transcribing the ds DNA with an RNAP to
form an RNA chimera. Examples of an RNA chimera might include part
or all of one or more the following: a guide RNA, an aptamer, a
mRNA, a tRNA, a microRNAs a shRNA, an snRNA, a short non-coding
RNA, a long non-coding RNA, an RNA probe, and a ribozyme.
[0018] In general, in one aspect a method is provided for making an
RNA guided protein that includes using the methods described herein
to make a single RNA polynucleotide derived from at least two DNA
oligonucleotides using the methods described above.
[0019] In general, in one aspect, a kit is provided that includes a
single oligonucleotide which is preferably a synthetic DNA
oligonucleotide and which has a sequence module at its 3' end that
is capable of hybridizing to an overlapping complementary 3'
sequence of a customer selected synthetic
[0020] DNA having a sequence which when transcribed into RNA is
capable of targeting a DNA sequence of interest. An RNA polymerase
specific promoter may be positioned within the single DNA
oligonucleotide. The kit may also contain a DNA polymerase capable
of extending the 3' end of the oligonucleotides to produce double
stranded DNA and also an RNA polymerase to transcribe the DNA
[0021] These and other features of the present teachings are set
forth herein.
BRIEF DESCRIPTION OF THE FIGURES
[0022] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way. FIG. 1A-1C shows a schematic representation of the assembly
and transcription in a single reaction of two ss DNA
oligonucleotides with modular sequences. An advantage of combining
a plurality of components is that one component may be varied and
the other remain constant so that using permutations and
combinations, it is possible to create a single designer RNA or
create a library of RNA molecules.
[0023] FIG. 1A shows two synthetic ss DNA oligonucleotides where
the first ss DNA oligonucleotide contains the promoter and partial
coding strand of a ds DNA that serves as a template for
transcription to form a chimeric RNA. The first ss DNA
oligonucleotide also has a sequence at the 3' end that is
complementary to the 3' end of a second oligonucleotide. The second
ss DNA oligonucleotide contains the 3' complementary sequence that
is part of the partial template strand. Extension of the first and
second ss DNA oligos by DNA polymerase creates a complete coding
strand and a complete template strand. The resulting ds DNA is the
template molecule for transcription of the chimeric RNA. The
"coding" strand and the "template strand" that form the ds DNA
template molecule are terms of art referring to the RNAP and
transcription. The coding strand may also be referred to as the
"top" strand" and has a sequence corresponding to all or part of
the final RNA transcript. The "template" strand may also be
referred to as the bottom strand and represent a sequence that is
complementary to all or part of the final RNA transcript.
[0024] FIG. 1B shows ds DNA formed by extension of the 3' ends of
each ss DNA oligonucleotide. The first oligonucleotide is the
template for the DNA polymerase that extends the 3' end of the
second oligonucleotide and vice versa. The region of the synthetic
DNA corresponding to the RNAP promoter becomes active for
transcription as soon as the ds DNA is formed.
[0025] FIG. 1C shows that the chimeric RNA polynucleotides are
transcribed rapidly and accurately by the RNAP from the template
strand in the DNA duplex. Transcription occurs as soon as the ds
promoter is formed in the reaction.
[0026] FIG. 2A-2F shows how the assembly reaction in FIG. 1 can be
applied to making custom guide RNA in a single reaction suitable
for activating a S. Pyogenes Cas9 protein.
[0027] FIG. 2A shows a first ss DNA oligonucleotide containing a
variable region inserted (which encodes a targeting region of a
single guide RNA (sgRNA)) between a T7 RNA promoter and a
complementary region for hybridizing with a second synthetic ss
oligonucleotide. The first ss DNA oligonucleotide includes a module
sequence for T7 RNAP promoter required for transcription of
adjacent DNA sequence. Also included is a variable sequence module
which is part of the coding strand of the final template. The
second ss DNA oligonucleotide includes a module sequence for
tracrRNA required for a functional single guide RNA.
[0028] FIG. 2B shows the ds DNA product of the 3' extension
reaction of both synthetic ss oligonucleotides that contains
sequences for the T7 RNA promoter, the variable region and the
tracrRNA.
[0029] FIG. 2C shows transcription of ds DNA to form a single guide
RNA. Transcription begins at the 3' end of the top strand of the T7
promoter.
[0030] FIG. 2D shows the products of the reaction detailed above
after treatment with DNAsel and purification. Products were
separated on a 2% agarose gel and photographed under UV
transillumination. The first lane contains an RNA molecular size
standard (Low range (LR) ss RNA). The second lane contains the
purified sgRNA (in vitro transcription (IVT) products) from the
reaction.
[0031] FIG. 2E and 2F show the specific ds DNA endonuclease
activity of S. pyogenes Cas9 ribonucleoprotein programmed with a
single guide RNA produced as outlined in FIG. 2A-2C.
[0032] In FIG. 2E, the substrate is a .about.514 bp PCR product
that is digested into 2 fragments of .about.336 and .about.178 bp
after incubation with sgRNA programmed Cas9 protein in a 2 step
reaction. Cas9 nuclease was programmed in vitro using purified
reaction products depicted in FIG. 2A-D. Cleavage reactions were
resolved on 1% agarose TBE gels, stained with ethidium bromide and
photographed under UV transillumination.
[0033] The first lane contains a ds DNA ladder (PCR marker) with
sizes as indicated.
[0034] The second lane contains target DNA incubated with Cas9
protein but no guide RNA (+Cas9/-sgRNA).
[0035] The third lane contains target DNA incubated with purified
sgRNA products detailed in Example 1 but no Cas nuclease
(-Cas9/+sgRNA).
[0036] The fourth lane contains target DNA incubated with Cas9
protein that was previously incubated with purified sgRNA products
as detailed in Example 1 (+Cas9/+sgRNA).
[0037] In FIG. 2F, the substrate is a .about.4361 bp linearized
plasmid DNA that is digested into 2 fragments of .about.2807 and
.about.1554 bp after incubation with sgRNA programmed Cas9 protein.
Cleavage reactions were resolved on 1% agarose TBE gels, stained
with ethidium bromide and photographed under UV
transillumination.
[0038] The first lane contains a ds DNA ladder with sizes as
indicated (1 kb ladder). The second lane contains target DNA
without digestion (uncut linear PBR322).
[0039] The third lane contains target DNA digested with S. pyogenes
Cas9 nuclease programmed in vitro using purified reaction sgRNA
products depicted in FIG. 2A-C.
[0040] The fourth lane contains target DNA digested with S.
pyogenes Cas9 nuclease within the template assembly and
transcription reaction as depicted in FIG. 2A-C and that also
contained recombinant Cas9 nuclease protein (one step Cas 9
digest). One step refers to the reaction mixture containing the Cas
nuclease while two step refers to addition of Cas nuclease to the
reaction mixture after synthesis of the guide RNA.
[0041] FIG. 3A-3D shows how the reaction in FIG. 1 can be applied
to making custom guide RNA in a single reaction suitable for
activating as N. meningitidis Cas9 protein.
[0042] FIG. 3A shows a first ss DNA oligonucleotide containing a
DNA targeting region (which corresponds to the targeting region of
a single guide RNA) inserted between a T7 RNA promoter and a region
for hybridizing with a second synthetic ss oligonucleotide. The
first ss DNA oligonucleotide contains the sequence for the top
strand of the T7RNAP promoter and a targeting region. The targeting
region is the sequence by which the final guide RNA associated with
Cas is directed to specific sequences in genomic DNA. The second ss
DNA oligonucleotide contains the sequence corresponding to N.
meningitidis tracrRNA in the sgRNA.
[0043] FIG. 3B shows that the extended oligonucleotides in FIG. 3A
provide a ds DNA that can serve as a transcription template for
sgRNA for N. meningitidis Cas9.
[0044] FIG. 3C shows the N. meningitidis Cas9 sgRNA transcription
products of the template assembly and transcription reaction.
[0045] FIG. 3D shows the transcription products of long and short
RNAs after treatment with DNAsel and subsequent purification.
Products were separated on a denaturing 6% polyacrylamide TBE Urea
gel, stained with SYBR Gold, and photographed under UV
transillumination.
[0046] The first lane contains an RNA molecular size standard.
[0047] The second lane contains the purified sgRNA from the
reaction using the short (96 nt) second template oligonucleotide,
and
[0048] The third lane contains the purified sgRNA from the reaction
using the longer (120 nt) second template oligonucleotide.
[0049] FIG. 3E shows the specific ds DNA endonuclease activity of
N. meningitidis Cas9 programmed with a single guide RNA after
addition of Cas nuclease to the reaction mixture containing
synthesized guide RNA. The method permitted rapid analysis of the
efficacy of short and long guide RNAs when combined with Cas for
endonuclease cleavage. The long and short sequences differed by a
3'-extension of the sgRNA. The substrate of the cleavage reaction
shown is a .about.550 bp PCR product that was digested into 2
fragments of .about.350 and .about.200 bp after incubation with
short or long sgRNA programmed Cas9 protein. Differing molar ratios
of Cas9 programmed with sgRNA: target are shown as indicated for
each target DNA (Nme WTAP Ex8) long (FL) and NmeWTAP Ex8 short (S))
reactions.
[0050] Digestion reactions were separated on 1.5% agarose TBE gel,
stained with ethidium bromide and photographed under UV
transillumination. Both long and short sgRNAs were shown to be
active. An advantage of this method is the ability to rapidly test
for active guide RNA sequences with little previous knowledge of
what sequences would be active.
[0051] FIG. 4A-4D shows how the reaction in FIG. 1 can be applied
to making custom guide RNA in a single reaction suitable for
activating a S. aureus Cas9 protein.
[0052] FIG. 4A is similar to FIG. 3A except the second
oligonucleotide contains a sequence module for S. aureus
tracrRNA.
[0053] FIG. 4B is similar to FIG. 3B except that S. aureus Tracr
RNA has become part of the duplex DNA template for transcription.
FIG. 4C shows the S. aureus Cas9 sgRNA transcription products of
the template assembly and transcription reaction.
[0054] FIG. 4D shows the specificity of cleavage of a ds DNA target
by S. aureus Cas9 ribonucleoprotein programmed with the single
guide RNA in FIG. 4A-4C.
[0055] In FIG. 4D, the substrate is a .about.2700 bp linearized
plasmid DNA that is digested into 2 fragments of .about.1300 after
incubation with sgRNA programmed Cas9 protein. Cleavage reactions
were resolved on 1% agarose TBE gels, stained with ethidium bromide
and photographed under UV transillumination.
[0056] The first lane contains the target DNA incubated alone.
[0057] The second lane contains a ds DNA ladder.
[0058] The third to sixth lanes contain target DNA incubated with
recombinant S. aureus Cas9 protein programmed with the products of
the reaction depicted in FIG. 4A-4C. The fold excess of S. aureus
Cas9/sgRNA to a constant amount of target DNA (3 nM target
DNA/lane) is shown above each lane.
[0059] FIG. 5A-5E shows custom DNA template assembly and
transcription of sgRNA for Acidaminococcus sp BV3L6 Cpf1 in a
single reaction, Cpf1 programming and target DNA digestion.
[0060] FIG. 5A schematically depicts the design of ss DNA
oligonucleotides for Cpf1 sgRNA template assembly and
transcription. Sequence modules on the first ss DNA oligonucleotide
include a T7 promoter, a constant region (tracrRNA) and a
complementary region (overlap) while the second ss DNA
oligonucleotide has a sequence module for targeting substrate
duplex DNA and a complementary sequence.
[0061] FIG. 5B depicts the fully extended ds DNA transcription
template.
[0062] FIG. 5C shows the Acidaminococcus sp Cas9 sgRNA
transcription products of the template assembly and transcription
reaction.
[0063] FIG. 5D shows the sgRNA produced in FIG. 5C after DNAseI and
purification. Products were separated on a denaturing 6%
polyacrylamide TBE Urea gel, stained with SYBR Gold, and
photographed under UV transillumination. The first lane contains an
RNA molecular size standard. The second lane contains the purified
sgRNA from the reaction.
[0064] In FIG. 5E, the substrate is a .about.2700 bp linearized
plasmid DNA that is digested into 2 fragments of .about.1300 bp
after incubation with sgRNA programmed Cas9 protein. Cleavage
reactions were resolved on 1% agarose TBE gels, stained with
ethidium bromide and photographed under UV transillumination. The
first lane contains a ds DNA ladder with sizes as indicated. The
second to sixth lanes contain target DNA incubated with recombinant
Acidaminococcus sp BV3L6 CPF1 protein programmed with the sgRNA
made as shown in FIG. 5A-5C. The fold excess of Acidaminococcus sp
BV3L6 CPF1/sgRNA to target DNA is shown above each lane.
[0065] FIG. 6A-6B shows how an arrayed library of multiple single
guide RNAs can be made in a highly parallel, high throughput,
multiwell plate based format.
[0066] FIG. 6A depicts a workflow for the synthesis of multiple
sgRNAs where (1) is design and manufacture of 96 ss DNA
oligonucleotides; (2) assembly of duplex DNA and transcription; (3)
cleanup of sample with DNase treatment and RNA cleanup; and (4) RNA
analysis.
[0067] FIG. 6B shows the distribution of RNA yields from an
experiment as outlined in FIG. 2A-2C where 96 individual synthetic
ss DNA oligonucleotides were designed and procured in a 96-well
plate. The 96 individual ss DNA oligonucleotides were combined with
a single sequence second ss DNA oligonucleotide in a separate
96-well plate under the reaction conditions described, then
purified. The yield of RNA for each of the arrayed library members
was determined by spectrophotometry. The results show that variable
sequences provide a range of yields under optimized conditions
described in the examples. Where yield is not required to be
optimized, conditions can be varied.
[0068] FIG. 7A-7D shows DNA template assembly and transcription of
a functional RNA aptamer. Here the Mango RNA aptamer in a scaffold
of 6S RNA is synthesized in a single reaction.
[0069] FIG. 7A shows the design of ss DNA oligonucleotide templates
for assembly and transcription.
[0070] FIG. 7B shows the fully extended transcription template
(duplex DNA).
[0071] FIG. 7C depicts the RNA products of the reaction.
[0072] FIG. 7D shows the activity of purified RNA mango made
according to FIG. 7A-7C. The mango RNA was mixed at the
concentrations provided above each lane (0, 0.5, 0.25, 0.125, 0.06
and 0.03 .mu.M) with buffer and TO1 fluorophore. Tubes were
photographed under UV transillumination. Brighter fluorescence is
seen as lighter color.
[0073] FIG. 8A-8D shows how a modular functional dimeric broccoli
aptamer RNA may be synthesized in a single reaction. Broccoli is a
49-nt aptamer. Dimeric broccoli is two broccoli aptamers within one
long stem loop in a backbone of a scaffold sequence F30 based on
the naturally occurring phi-29 viral RNA three-way junction motif.
diBroccoli was inserted into each of two entry points in F30 to
create F30-2xdBroccoli. Thus, this F30 scaffold presents the
equivalent of four Broccoli units and is roughly four times
brighter than F30-Broccoli (Svensen, et al., Cell Chemical Biology,
23:415-25, 2016).
[0074] FIG. 8A shows the design of first and second ss DNA
oligonucleotides where the first ss DNA oligonucleotide contains a
sequence module for a T7 promoter and the second ss oligonucleotide
contains two Broccoli sequence modules with a spacer between.
Complementary sequences are present at the 3' end of both
oligonucleotides.
[0075] FIG. 8B shows the fully extended duplex DNA transcription
template.
[0076] FIG. 8C shows the RNA transcription products.
[0077] FIG. 8D shows the activity of functional 2x diBroccoli RNA.
The RNA produced according to
[0078] FIG. 8A-8C was purified, and mixed at the concentrations
shown (0, 0.5, 0.25, 0.125, 0.06 and 0.03 .mu.M) with buffer and
DHBF1 fluorphore. Tubes were scanned on a Typhoon multi-imager with
excitation at 457 nm and emission detected at 526 nm. Brighter
fluorescence is seen as darker color.
[0079] FIG. 9 shows the RNA products obtained from Example 6 and 7.
Reaction products from template assembly and transcription
reactions were separated by denaturing polyacrylamide gel
electrophoresis, stained with SYBR Gold, and photographed under UV
transillumination. The first lane contains an RNA size marker, the
second lane contains Broccoli reaction products from Example 7. The
third lane contains Mango reaction products from Example 6.
DETAILED DESCRIPTION OF EMBODIMENTS
[0080] Unless defined otherwise herein, all technical and
scientific terms used herein have the same meaning as commonly
understood by one of ordinary skill in the art to which this
invention belongs. Although any methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of the present invention, the preferred methods and
materials are described.
[0081] All patents and publications, including all sequences
disclosed within such patents and publications, referred to herein
are expressly incorporated by reference.
[0082] Numeric ranges are inclusive of the numbers defining the
range. Unless otherwise indicated, nucleic acids are written left
to right in 5' to 3' orientation; amino acid sequences are written
left to right in amino to carboxy orientation, respectively.
[0083] The headings provided herein are not limitations of the
various aspects or embodiments of the invention. Accordingly, the
terms defined immediately below are more fully defined by reference
to the specification as a whole.
[0084] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0085] Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR
BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale
& Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper
Perennial, N.Y. (1991) provide one of skill with the general
meaning of many of the terms used herein. Still, certain terms are
defined below for the sake of clarity and ease of reference.
[0086] The term "oligonucleotide" as used herein denotes a ss
multimer of nucleotide of from about 2 to 200 nucleotides, up to
500 nucleotides in length. Oligonucleotides may be synthetic or may
be made enzymatically, and, in some embodiments, are 30 to 150
nucleotides in length. Oligonucleotides may contain ribonucleotide
monomers (i.e., may be oligoribonucleotides) or deoxyribonucleotide
monomers, or both ribonucleotide monomers and deoxyribonucleotide
monomers. An oligonucleotide may be 10 to 20, 11 to 30, 31 to 40,
41 to 50, 51-60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150
to 200 nucleotides in length, for example.
[0087] The term "mixture", as used herein, refers to a combination
of elements, that are interspersed and not in any particular order.
Examples of mixtures of elements include a number of different
elements that are dissolved in the same aqueous solution. A mixture
is not addressable. To illustrate by example, an array of spatially
separated surface-bound polynucleotides, as is commonly known in
the art, is not a mixture of surface-bound polynucleotides because
the species of surface-bound polynucleotides are spatially
distinct, and the array is addressable.
[0088] The term "duplex," or "duplexed," as used herein, describes
two complementary polynucleotides that are base-paired, i.e.,
hybridized together.
[0089] A "plurality" contains at least 2 members. In certain cases,
a plurality may have at least 10, at least 100, at least 100, at
least 10,000, at least 100,000, at least 10.sup.6, at least
10.sup.7, at least 10.sup.8 or at least 10.sup.9 or more
members.
[0090] If two nucleic acids are "complementary", they hybridize
with one another under high stringency conditions. The term
"perfectly complementary" is used to describe a duplex in which
each base of one of the nucleic acids base pairs with a
complementary nucleotide in the other nucleic acid. In many cases,
two sequences that are complementary have at least 10, e.g., at
least 12, at least 15 or at least 20 nucleotides of
complementarity.
[0091] The term "strand" as used herein refers to a nucleic acid
made up of nucleotides covalently linked together by covalent
bonds, e.g., phosphodiester bonds. In a cell, DNA usually exists in
a ds form, and as such, has two complementary strands of nucleic
acid referred to herein as the "top" and "bottom" strands. In
certain cases, complementary strands of a chromosomal region may be
referred to as "plus" and "minus" strands, the "first" and "second"
strands, the "coding" and "noncoding" strands, the "Watson" and
"Crick" strands or the "sense" and "antisense" strands. The
assignment of a strand as being a top or bottom strand is arbitrary
and does not imply any particular orientation, function or
structure.
[0092] The term "hybridizing" or "hybridizes" refers to a process
in which a nucleic acid strand anneals to and forms a stable
duplex, either a homoduplex or a heteroduplex, under normal
hybridization conditions with a second complementary nucleic acid
strand, and does not form a stable duplex with unrelated nucleic
acid molecules under the same normal hybridization conditions. The
formation of a duplex is accomplished by annealing two
complementary nucleic acid strands in a hybridization reaction. The
hybridization reaction can be made to be highly specific by
adjustment of the hybridization conditions (often referred to as
hybridization stringency) under which the hybridization reaction
takes place, such that hybridization between two nucleic acid
strands will not form a stable duplex, e.g., a duplex that retains
a region of double strandedness under normal stringency conditions,
unless the two nucleic acid strands contain a certain number of
nucleotides in specific sequences which are substantially or
completely complementary.
[0093] The term "extending", as used herein, refers to the
extension of a nucleic acid, e.g., a primer or a primer extension
product, by the addition of nucleotides using a polymerase. For
example, if a primer that is annealed to a nucleic acid is
extended, the nucleic acid acts as a template for extension
reaction.
[0094] As used herein, the term "overlapping sequence", refers to a
sequence that is complementary in two polynucleotides and where the
overlapping sequence is ss, on one polynucleotide it can be
hybridized to another overlapping complementary ss region on
another polynucleotide. By way of example, the overlapping sequence
may be complementary in at least 5, 10, 15, or more polynucleotides
in a set of polynucleotides. An overlapping sequence may be at or
close to (e.g., within about 5, 10, 20 nucleotides of) the 3' ends
of two distinct molecules (e.g., the 3' ends of two ss
oligonucleotides, or the 3' end of the top strand of first ds
polynucleotide and the 3' end of the bottom strand of a second ds
molecule), where, if the non-overlapping sequence is at the 3' ends
then the non-overlapping sequence may be removed using a 3'-5'
exonuclease activity of a polymerase. An overlapping sequence may
vary in length and, in some cases, may be at least 12 nucleotides
in length (e.g. at least 15, 20 or more nucleotides in length) and/
or may be up 100 nucleotides in length (e.g., up to 50, up to 30,
up to 20 or up to 15 nucleotides in length).
[0095] As used herein, the term "buffering agent", refers to an
agent that allows a solution to resist changes in pH when acid or
alkali is added to the solution. Examples of suitable non-naturally
occurring buffering agents that may be used in the compositions,
kits, and methods of the invention include, for example, Tris,
HEPES, TAPS, MOPS, tricine, or MES.
[0096] The term "non-naturally occurring" refers to a composition
that does not exist in nature.
[0097] Any protein described herein may be non-naturally occurring,
where the term "non-naturally occurring" refers to a protein that
has an amino acid sequence and/or a post-translational modification
pattern that is different to the protein in its natural state. For
example, a non-naturally occurring protein may have one or more
amino acid substitutions, deletions or insertions at the
N-terminus, the C-terminus and/or between the N- and C-termini of
the protein. A "non-naturally occurring" protein may have an amino
acid sequence that is different to a naturally occurring amino acid
sequence (i.e., having less than 100% sequence identity to the
amino acid sequence of a naturally occurring protein) but that that
is at least 80%, at least 85%, at least 90%, at least 95%, at least
97%, at least 98% or at least 99% identical to the naturally
occurring amino acid sequence. In certain cases, a non-naturally
occurring protein may contain an N-terminal methionine or may lack
one or more post-translational modifications (e.g., glycosylation,
phosphorylation, etc.) if it is produced by a different (e.g.,
bacterial) cell. A "mutant" protein may have one or more amino acid
substitutions relative to a wild-type protein and may include a
"fusion" protein. The term "fusion protein" refers to a protein
composed of a plurality of polypeptide components that are unjoined
in their native state. Fusion proteins may be a combination of two,
three or even four or more different proteins. The term polypeptide
includes fusion proteins, including, but not limited to, a fusion
of two or more heterologous amino acid sequences, a fusion of a
polypeptide with: a heterologous targeting sequence, a linker, an
immunologically tag, a detectable fusion partner, such as a
fluorescent protein, B-galactosidase, luciferase, etc., and the
like. A fusion protein may have one or more heterologous domains
added to the N-terminus, C-terminus, and or the middle portion of
the protein. If two parts of a fusion protein are "heterologous",
they are not part of the same protein in its natural state.
[0098] In the context of a nucleic acid, the term "non-naturally
occurring" refers to a nucleic acid that contains: a) a sequence of
nucleotides that is different to a nucleic acid in its natural
state (i.e. having less than 100% sequence identity to a naturally
occurring nucleic acid sequence), b) one or more non-naturally
occurring nucleotide monomers (which may result in a non-natural
backbone or sugar that is not G, A, T or C) and/or c) may contain
one or more other modifications (e.g., an added label or other
moiety) to the 5'-end, the 3' end, and/or between the 5'- and
3'-ends of the nucleic acid.
[0099] In the context of a preparation, the term "non-naturally
occurring" refers to: a) a combination of components that are not
combined by nature, e.g., because they are at different locations,
in different cells or different cell compartments; b) a combination
of components that have relative concentrations that are not found
in nature; c) a combination that lacks something that is usually
associated with one of the components in nature; d) a combination
that is in a form that is not found in nature, e.g., dried, freeze
dried, crystalline, aqueous; and/or e) a combination that contains
a component that is not found in nature. For example, a preparation
may contain a "non-naturally occurring" buffering agent (e.g.,
Tris, HEPES, TAPS, MOPS, tricine or MES), a detergent, a dye, a
reaction enhancer or inhibitor, an oxidizing agent, a reducing
agent, a solvent or a preservative that is not found in nature.
[0100] Any of the enzyme listed herein may have an amino acid
sequence that is identical to that of a naturally occurring enzyme,
or a sequence that is at least 90% identical, e.g., at least 95%
identical, to a naturally occurring enzyme.
[0101] Compositions and methods are provided herein for assembling
polynucleotides from synthetic oligonucleotides in a single
reaction vessel. Assembly of polynucleotides is fundamentally
different from amplification of naturally occurring target
polynucleotides that occur in nature within a mixture that includes
non-target DNAs. In amplification, multiple rounds of
oligonucleotide primer extension (in the case of DNA amplification)
occur in a reaction in order to increase the quantity of or to
create additional copies of a starting molecule (template) or
portion of a starting molecule.
[0102] In embodiments of the invention, assembly as used herein
refers to the hybridization of oligonucleotide substrates in the
reaction to create a template for DNA polymerase for 3' extension
of the ends of the oligonucleotides. The assembled template is a ds
DNA with an RNAP promoter that can be transcribed into a novel
non-natural RNA that is not encoded by the sequence of the
individual input DNA oligonucleotides.
[0103] No denaturation step is required, because the inputs are
less complex than a mixture of genomic DNA, or cDNA prepared from
cellular total or mRNA prepared using random priming, oligo d(T)
priming or other cDNA synthesis priming strategies that produce
complex mixtures of cDNA. The oligonucleotide templates are
designed so that annealing occurs efficiently at room
temperature.
[0104] No DNA amplification takes place in the method, and no
purified genomic DNA, or cDNA prepared from RNA derived from
biological sources is used. Instead, the method uses relatively
small, synthetic DNA oligonucleotide that do not occur in nature
and that contain functional sequence modules that may or may not be
derived from nature to assemble larger purpose-designed
polynucleotides.
[0105] One feature of present embodiments is the use of an RNAP
promoter and RNAP such as any RNAP and promoter known in the art.
Certain examples herein not intended to be limiting, describe the
use of T7 RNAP and T7 RNAP promoter. Other examples of polymerases
with promoters include T7 RNAP with T7 Class III RNAP promoter or
T7 phi 2.5 RNAP promoter, SP6 RNAP with SP6 RNAP promoter, T3 RNAP
with T3 RNAP promoter, Syn5 RNAP with Syn5 RNAP promoter, E. coli
RNAP with T5 promoter, E. coli RNAP with a standard E. coli
promoter that is active in vitro e.g. TTGACAN(17)TATAAT (SEQ ID
NO:1), or Tac promoter and promoters recognized by thermostable
RNAPs (New England Biolabs, Ipswich, Mass.). Commonly used phage
RNAPs for use herein are usually specific for promoters in their
genomes. However, some phage promoters use host RNAP. See above
example of T5 promoter with E. coli RNAP.
[0106] One feature of present embodiments is a DNA polymerase.
Embodiments utilize mesophilic DNA polymerases such as T4 DNA
polymerase. E. coli DNA polymerase I Klenow fragment exo minus, and
E. coli DNA polymerase I Klenow fragment. Other polymerases include
Family B DNA polymerases such as Pfu DNA polymerase, Q5, Phusion,
Family A DNA polymerases such as E. coli DNA polymerase I, Taq, and
Taq variants including HemoKlentaq, Bst DNA polymerase, and strand
displacing DNA polymerases such as Sulphololobus, and Bst variants
such as Bst 2.0, Bst 3.0, or Phi29 (the specific identified
polymerases are available from New England Biolabs, Ipswich,
Mass.).
[0107] The design of the synthetic ss DNA oligonucleotides requires
an overlapping region of complementary bases. The number of
nucleotides in an overlapping region is in the range of at least 5
nucleotides to as long as practical because of cost constraints.
For example, embodiments include ranges of lengths of overlapping
sequences include 5 nucleotides (nt) -50 nt or 5 nt-40 nt, or 5
nt-35 nt, or 5 nt-30 nt or 5 nt-25 nt.
[0108] The size of ss oligonucleotides fragments for joining to
form a long polynucleotide is limited only by the length of
oligonucleotide that is cost efficient to synthesize in the upper
range and by a size suited to incorporate an RNAP promoter and a
region of overlap in the lower range. For example, a single
oligonucleotide might be 20-500 nts in length for example 20-200
nts for example 20-150 nts in length.
[0109] Once joined, the newly synthesized RNA may be of any desired
size. In a preferred embodiment, the shortest synthetic fragment
made from two oligonucleotides would be 18 nt and the largest would
be approximately double the maximum size of an oligonucleotide that
is capable of being synthesized in a cost effective manner for
example, about 1000 nt. Examples include 18 nt-393 nt or 25 nt-350
nt or 40 nt-300 nt or 60 nt-260 nt. Multimers of synthetic
fragments can be joined by ligation or other means to form a
polynucleotide of any desired length.
[0110] Examples of RNA that can be synthesized by the present
methods or using the present compositions include guide RNA,
aptamers, mRNAs, tRNAs, microRNAs, shRNAs, snRNAs, small non-coding
RNAs, RNA probes, ribozymes or any other type of RNA as
desired.
[0111] In embodiments of the invention, all the reagents for
assembling a single DNA molecule from synthetic oligonucleotides
and also for transcription of the DNA molecules into RNA molecules
are provided in a single reaction mixture.
[0112] In embodiments of the invention, guide RNA can be made from
joined and transcribed ss synthetic oligonucleotides in a single
reaction mixture that additionally includes the Cas enzyme to which
the guide RNA attaches to enable the complex to act as a sequence
specific nuclease in the presence of a DNA target additionally
added to the reaction mixture. We demonstrated this by showing
cleavage of template DNA resulting from incorporation of sgRNA into
Cas9 (programming) in Example 1 and FIG. 2A-2F.
[0113] This is significant because the method markedly shortens
current workflows and reduces expense for in vitro programming of
Cas9. This is achieved by using inexpensive and rapidly obtainable
ss DNA oligonucleotides as substrate inputs as compared to
commercially assembled ds DNA constructs such as gBlocks (IDT,
Coralville, Iowa) or gene-strings (Life Technologies, Carlsbad,
Calif.) which take longer to obtain and may require amplification
because they are supplied at low concentration. As compared to
producing plasmid DNA templates for sgRNA synthesis, the workflow
of the current method is much shorter. Finally, in contrast to
costly and not rapidly obtainable commercially synthesized RNA
oligonucleotides (IDT, GE Dharmacon, Lafayette, Colo.), the ss DNA
oligonucleotides used herein are significantly less costly and more
efficient to obtain for commercial use.
[0114] The programming of Cas9 nuclease within the sgRNA template
assembly and transcription reaction is significant because this
allows for the rapid and inexpensive creation of sequence-specific
ds DNA endonucleases in a one-step reaction by adding a relatively
short, inexpensive, and readily available custom ss DNA
oligonucleotide. In essence, this method allows one to create a
custom endonuclease capable of cutting almost any sequence and
suitable for recombinant DNA manipulation (e.g. molecular cloning
of DNA fragments) in minutes.
[0115] For sgRNA programming of Cas9 or Cas9 ortholog proteins, a
target region denotes a specific nucleic acid sequence to which a
Cas nuclease is directed for cutting, nicking or binding that is
determined by the sequence of the guide RNA and protospacer
adjacent motif (PAM) sequence requirements of the Cas9 or Cas9
ortholog protein.
[0116] Assembly of a plurality of oligonucleotides into a duplex
DNA that can be transcribed to a single RNA in a single reaction
chamber and in a single reaction step has many uses. For example,
it is possible to assemble large RNA molecules from more than two
ss oligonucleotides. In the figures and examples provided herein,
some sequence modules are described on the first ss DNA
oligonucleotide and other sequence modules are described on the
second ss DNA oligonucleotide. However, the assembly method is not
dependent on the order in which the sequence modules occur. They
might be switched around as determined to be optimal by the
intended use. Indeed, in FIG. 5A-5E the targeting sequence module
is on the second ss DNA oligonucleotide whereas in FIG. 4A-4D, the
targeting sequence module is on the first ss oligonucleotide.
[0117] Single RNA molecules of varying lengths may be transcribed
from assembled duplex DNA where the limitation of length of ss DNA
oligonucleotides is limited only by the cost and efficiency of
synthesis of the oligonucleotides,
[0118] Alternatively, 4 oligonucleotides or more generally 2
oligonucleotides may be assembled in which the pairs of
oligonucleotides may become assembled after hybridization of
complementary sequences at their 3' ends. In addition, the second
oligonucleotide may hybridize to a third oligonucleotide by
complementary sequences at the 5' end. A strand displacing
polymerase may read through the third and fourth ss DNA
oligonucleotide resulting in an extended duplex DNA and when
transcribed are large RNA. Alternatively, a non strand displacing
polymerase may be utilized to complete the duplex where a ligase is
added to close nicks.
[0119] Because any sequence can be designed for ss DNA
oligonucleotide synthesis, it is possible to design sequence
modules which may be characterized by their function within the ss
DNA oligonucleotide sequence. The sequence modules may be fixed for
a population of ss DNA oligonucleotides or may contain sequences
that vary by design or randomly with the population. In this way,
assembled DNA duplexes and transcribed RNA may represent a diverse
population wherein members have variable and fixed sequence
modules, and may be of any desired length.
[0120] One application of the described method of assembling duplex
DNA and transcribing RNA in a single step reaction is to generate
guide RNAs. Another application is to generate RNA aptamers that
may react with a detectable marker or may react with another
sequence module to generate a signal. This is illustrated herein
with mango and broccoli aptamers. These aptamers are suited for
demonstrating the utility of the method as they readily bind a
detector dye. However, the assembly method described herein may be
generally applicable to other biological markers that involve RNA
molecules.
[0121] In some embodiments, it may be desirable to immobilize one
or more of the ss DNA oligonucleotides or the transcribed RNA
before, during or after the assembly reaction. Immobilization may
occur by means of coated or uncoated beads, microwell dishes,
columns, papers, microfluidic devices etc. Immobilization may occur
through affinity binding of the DNA or RNA by means of a modified
nucleotide or biotin/streptavidin binding or other affinity binding
molecule.
Kits
[0122] Also provided by this disclosure is a kit for practicing the
subject method, as described above. A kit may contain any
combination of the reagents described above, e.g.,
oligonucleotides, dNTPs, riboNTPs, DNA polymerase, RNA polymerase,
etc. The various components may be in different containers or in
the same container.
[0123] In addition, the kit may also comprise reagents for
performing the reaction, e.g., one or more buffers. The various
components of the kit may be present in separate containers or
certain compatible components may be pre-combined into a single
container, as desired.
[0124] In addition to above-mentioned components, the subject kits
may further include instructions for using the components of the
kit to practice the subject methods, i.e., to provide instructions
for sample analysis. The instructions for practicing the present
method may be recorded on a suitable recording medium. For example,
the instructions may be printed on a substrate, such as paper or
plastic, etc. As such, the instructions may be present in the kits
as a package insert, in the labeling of the container of the kit or
components thereof (i.e., associated with the packaging or
subpackaging) etc. In other embodiments, the instructions are
present as an electronic storage data file present on a suitable
computer readable storage medium, e.g., CD-ROM, diskette, etc. In
yet other embodiments, the actual instructions are not present in
the kit, but means for obtaining the instructions from a remote
source, e.g., via the internet, are provided. An example of this
embodiment is a kit that includes a web address where the
instructions can be viewed and/or from which the instructions can
be downloaded. As with the instructions, this means for obtaining
the instructions is recorded on a suitable substrate.
[0125] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference. This includes U.S. patent application
Ser. No. 15/469,681, filed Mar. 27, 2017 and U.S. provisional
application Ser. No. 62/317,035 filed Apr. 1, 2016.
EXAMPLES
[0126] Aspects of the present teachings can be further understood
in light of the following examples, which should not be construed
as limiting the scope of the present teachings in any way.
Description of Terms
[0127] Clustered Regularly Interspersed Short Palindromic Repeats
Array (CRISPR).
[0128] CRISPR RNA (crRNA): A small RNA arising from transcription
and processing of a CRISPR array. crRNA interacts with tracrRNA and
Cas9 to direct the sequence specific binding of the complex.
[0129] Trans-activating crRNA (tracrRNA): A small non-coding RNA
transcribed from the CRISPR locus. The tracrRNA base pairs with a
crRNA. The crRNA and tracrRNA complex is incorporated into Cas9
proteins to enable cleavage of target sequences
[0130] Single guide RNA (sgRNA): A synthetic fusion of a crRNA and
tracrRNA, often connected by a GNRA tetraloop sequence that
provides both targeting specificity and scaffolding/binding ability
for Cas9 nuclease in a single polynucleotide (Jinek et al. (2012)
Science, 337(6096):816-21).
[0131] Cas: CRISPR-associated genes.
[0132] Cas9, Csn1: a CRISPR-associated protein containing two
nuclease domains, that is programmed by small RNAs to cleave
DNA
[0133] Nuclease-deficient Cas9 (dCAS9).
[0134] Cpf1: A naturally occurring two-component RNA programmable
DNA nuclease (class 2) lacking tracrRNA, associated with CRISPR. It
utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1
cleaves DNA via a staggered DNA ds break. It is obtainable from
Acidaminococcus.
[0135] Template refers to DNA that is used for RNA synthesis.
[0136] All reagents can be obtained commercially from New England
Biolabs, Ipswich, Mass. unless stated otherwise. Exemplary
conditions for assembly reactions of two oligonucleotides used in
the present examples
[0137] In some cases, the reaction may be done in a single closed
vessel, without changing the conditions of the reaction, opening
the vessel or adding additional reagents during the course of the
reaction.
Example 1. Custom S. pyogenes sgRNA DNA Template Assembly and
Transcription in a Single Reaction and Cas9 Programming in a Single
Reaction.
[0138] A 49 nt ss DNA oligonucleotide synthesized from three
functional modules: (a) the top strand of the T7 RNAP promoter; (b)
coding strand of a 20 nt region corresponding to a targeting
region, of an sgRNA; and (c) a region of 9 nt corresponding to the
repeat region of the S. pyogenes CRISPR repeat was added to a
reaction containing a second ss DNA oligonucleotide corresponding
to the template strand of the S. pyogenes tracrRNA connected to the
remainder of the S. pyogenes CRISPR repeat by a GAAA tetraloop
sequence (FIG. 2A) (Jinek, et al. (2012) Science,
337(6096):816-21). The first and second ss DNA oligonucleotides
were complementary to each other for 9 bp at their 3'ends.
[0139] The reaction was carried out by combining the
oligonucleotides, enzymes, and buffers as follows:
[0140] (a) 0.62 units (0.05-5 units) E. coli DNA polymerase I
Klenow fragment; 0.25 uM first synthetic or natural polynucleotide
and 0.25 uM second synthetic or naturally occurring polynucleotide
although oligonucleotides may be added in at any convenient
concentration, 33 um (1-100 micromolar) dNTP, T7 RNAP and buffers
such as provided by NEB in its HiScribe.TM. kit (New England
Biolabs, Ipswich, Mass.). Incubation in the core reaction mixture
was 37.degree. C. for 30 minutes for convenience although as little
as 1 minute or as much as 4 hours or up to overnight incubation can
be used.
[0141] (b) Cas9 capable of reacting with sgRNA to form a complex
with nuclease activity.
[0142] (c) Addition of target DNA for nuclease cleavage
[0143] (a), (b) and (c) can be performed separately (Example
1-6).
[0144] (a), and (b) can be carried out together in one tube and in
a single incubation followed by (c).
[0145] (a), (b) and (c) can be carried out together in one tube and
in a single incubation (Example 1).
[0146] Template assembly occurred via annealing of the 2
oligonucleotides and their extension by E. coli DNA polymerase I
Klenow fragment (FIG. 2B). Transcription was initiated as ds DNA
was formed in the reaction (FIG. 2C). sgRNA was purified, analyzed
by gel electrophoresis (FIG. 2D) and used for programming
recombinant Cas9 nuclease (FIG. 2E) where Cas9 was combined with
sgRNA in a second step to cleave a target DNA.
[0147] To assess the function of the sgRNA synthesized by the
method, equimolar sgRNA was mixed with Cas9 nuclease (30 nM final
concentration) in Cas9 reaction buffer (NEB 20 mM HEPES pH 6.5, 100
mM NaCl, 5 mM MgCl.sub.2, 0.1 mM EDTA) for 10 minutes at 25.degree.
C. Target DNA (3 nM 514 bp PCR product) was added followed by a
further incubation at 60 minutes at 37.degree. C. The results are
shown in FIG. 2E. When Cas9 was added to the assembly and
transcription reaction mixture at the outset, an excess of guide
RNA was generated by the synthesis reaction for programming Cas9
nuclease directly in the reaction mixture described above which
also contained target DNA (3 nM linearized PBR322). The results are
shown in FIG. 2F which demonstrated that template assembly,
transcription, loading of sgRNA into Cas9 protein, and cleavage of
target DNA occurred in the single reaction.
Example 2. Custom N. meningitidis sgRNA DNA Template Assembly and
Transcription in a Single Reaction
[0148] A 60 nt ss DNA oligonucleotide containing three sequence
modules: (a) the first strand of the T7 RNAP promoter; (b) coding
strand of a 22 nt region corresponding to a targeting region, of an
sgRNA; and (c) a region of 15 nt corresponding to the repeat region
of the N. meningitidis CRISPR repeat; was added to a reaction
containing a second ss DNA oligonucleotide corresponding to the
template strand of the N. meningitidis tracrRNA connected to the
remainder of the N. meningitidis CRISPR repeat by a GAAA tetraloop
sequence (FIG. 3A) (Esvelt KM, et al. (2013), Nature Methods 10:
1116-1121). The first and second ss DNA oligonucleotides were
complementary to each other for 15 bp at their 3'-ends. Long (120
nt) and short (96 nt) versions of the second oligonucleotide were
used to make long or short versions of the sgRNA. The long and
short sgRNAs differed by a 3'-exension of their sequence.
[0149] The oligonucleotides were combined with enzymes, and buffer
as described in Example 1 in a reaction vessel and incubated at
37.degree. C. for 30 minutes. Template assembly occurred via
annealing of the 2 oligonucleotides and their extension by E. coli
DNA polymerase I Klenow fragment (FIG. 3B). Transcription began as
ds DNA was formed in the reaction (FIG. 3C). sgRNA was purified,
analyzed by gel electrophoresis (FIG. 3D) and used for programming
recombinant Cas9 nuclease (FIG. 3E).
[0150] To assess the function of sgRNA synthesized by the method,
the sgRNA was mixed with recombinant N. meningitidis Cas9 nuclease
in Cas9 reaction buffer (NEB, 20 mM HEPES pH 6.5, 100 mM NaCl, 5 mM
MgCl.sub.2, 0.1 mM EDTA) for 10 minutes at 25.degree. C. and
substrate DNA was added followed by a further incubation at 60
minutes at 37.degree. C. The results are shown in FIG. 3E.
[0151] The results showed that the assembly method can be used for
the rapid production of sgRNAs for Cas9 orthologs that are not
commercially available or in wide use. In this case both the long
and short versions of the sgRNA synthesized supported the specific
ds DNA endonuclease activity of N. meningitidis Cas9.
Significantly, the approach described here will be useful in cases
where the sequences of the regions required for Cas9 ortholog
function are not known and where rapid prototyping of sgRNA
sequence and structure are desirable.
Example 3. Custom S. aureus sgRNA DNA template assembly and
transcription in a single reaction.
[0152] A 58 nt ss DNA oligonucleotide containing three functional
modules: (a) the first strand of the T7 RNAP promoter, (b) the
coding strand of a 22 nt region corresponding to a targeting region
of an sgRNA; and (c) a region of 15 nt corresponding to the repeat
region of the S. aureus CRISPR repeat; was added to a reaction
containing a second ss DNA oligonucleotide that was 77 nt in
length, corresponding to the template strand of the S. aureus
tracrRNA connected to the remainder of the S. aureus CRISPR repeat
by a GAAA tetraloop sequence (FIG. 4A). The first and second ss DNA
oligonucleotides were complementary to each other for 15 bp at
their 3' ends.
[0153] Reactions were carried out by combining the
oligonucleotides, enzymes, and buffers as described and incubating
at 37.degree. C. for 30 minutes. Template assembly occurred via
annealing of the 2 oligonucleotides and their extension by E. coli
DNA polymerase I Klenow fragment (FIG. 4B). Transcription began as
ds DNA is formed in the reaction (FIG. 4C). sgRNA was purified and
used for programming recombinant Cas9 nuclease (FIG. 4D).
[0154] To assess the function of sgRNA synthesized by the method,
the sgRNA was mixed with recombinant S. aureus Cas9 nuclease in
Cas9 reaction buffer (NEB, 20 mM HEPES pH 6.5, 100 mM NaCl, 5 mM
MgCl.sub.2, 0.1 mM EDTA) for 10 minutes at 25.degree. C. and
substrate DNA was added followed by a further incubation at 60
minutes at 37.degree. C. The results are shown in FIG. 4D.
[0155] This is another example of how the method can be used for
the rapid production of sgRNAs for Cas9 orthologs other than S.
pyogenes that are not commercially available or in wide use and for
which synthetic RNA oligonucleotides corresponding to tracr and
crRNAs, or other transcription templates are not commercially or
readily available.
Example 4. Custom Acidaminococcus sp BV3L6 CPF1 sgRNA DNA Template
Assembly and Transcription in a Single Reaction.
[0156] A 43 nt ss DNA oligonucleotide containing two functional
modules: (a) top strand of the T7 RNAP promoter, and (b) a coding
strand of a 20 nt region corresponding to a direct repeat of the
Acidaminococcus sp BV3L6 Cpf1 CRISPR repeat was added to a reaction
containing a second ss DNA oligonucleotide 35 nt in length and
corresponding to the template strand of the partial Acidaminococcus
sp BV3L6 Cpf1 CRISPR repeat and a 20 nt targeting region. The 3'
ends of the first and second ss DNA oligonucleotides were
complementary for 15 bp, and together form a template for a
complete Acidaminococcus sp BV3L6 Cpf1 sgRNA (FIG. 5A) (Zetsche et
al. (2015) Cell. 163: 1-22). No GAAA tetraloop linker was required
as the Cpf1 guide RNAs are single polynucleotides.
[0157] Reactions were carried out by combining the
oligonucleotides, enzymes, and buffers as described and incubating
at 37.degree. C. for 30 minutes. Template assembly occurred via
annealing of the 2 oligonucleotides and their extension by E. coli
DNA polymerase I Klenow fragment (FIG. 5B). Transcription began as
ds DNA was formed in the reaction (FIG. 5C). sgRNA was purified,
analyzed by gel electrophoresis (FIG. 5D) and used for programming
recombinant Cpf1 nuclease (FIG. 5E).
[0158] To assess the function of sgRNA synthesized by the method,
the sgRNA was mixed with recombinant Acidaminococcus sp BV3L6 Cpf1
nuclease in NEBuffer 4 (New England Biolabs, Ipswich, Mass.) for 10
minutes at 25.degree. C. and substrate DNA was added followed by a
further incubation at 60 minutes at 37.degree. C. The results are
shown in FIG. 5E.
[0159] This example demonstrates that the method is versatile and
can be used for the rapid production of sgRNAs for Cpf1 type
RNA-guided nucleases. Furthermore, the method can be used in cases
where the targeting region of the resulting sgRNA is non-adjacent
to the RNAP promoter and where synthetic RNA oligonucleotides
useful as guide RNAs, or other transcription templates are not
commercially or readily available.
Example 5. Synthesis of an arrayed S. pyogenes Cas9 sgRNA library
using DNA template assembly and transcription in parallel
reactions.
[0160] An arrayed library of S. pyogenes Cas9 sgRNAs was generated
as follows: A workflow is shown in FIG. 6A. 96 distinct 54-56 nt ss
DNA oligos were obtained from IDT (Coralville, Iowa) and arrayed in
a 96-well plate (one oligonucleotide per well). The ss DNA
oligonucleotides correspond to the top strand of the T7 RNAP
promoter, coding strand of a 20 nt region which differ in each of
the 96 oligonucleotides (taking the place of the targeting region
of an sgRNA) and a region of 14 nt complementary to the repeat
region of the S. pyogenes CRISPR repeat, and were added to a
reactions containing a second ss DNA oligonucleotide, common to
each of the 96 reactions, corresponding to the template strand of
the S. pyogenes tracrRNA connected to the remainder of the S.
pyogenes CRISPR repeat connected by a GAAA tetraloop sequence (FIG.
6A-6B).
[0161] Reactions were carried out in wells of a 96-well plates by
combining the oligonucleotides, enzymes, and buffers as described
and incubating at 37.degree. C. for 30 minutes. Template assembly
occurred via annealing of the 2 oligonucleotides and their
extension by E. coli DNA polymerase I Klenow fragment.
Transcription began as ds DNA is formed in the reaction.
[0162] Each member of the RNA library resulting from transcription
reactions was treated with DNasel, purified, and yield measured and
shown in FIG. 6B.
[0163] This example is significant because it demonstrates the
utility of the method for rapidly and inexpensively creating
arrayed libraries of sgRNAs for use in screening. Libraries could
have as few as 2 members, and as many as practical restraints would
allow. For example, the number of ss DNA oligonucleotide inputs, or
subsequent transfection or in vitro assays. For example, 96-well,
384-well, or 1536-well plates.
Example 6. Synthesis of functional RNA mango aptamers using DNA
template assembly and transcription in single reactions
[0164] RNA Mango is a 29 nt guanosine quadruplex containing RNA
aptamer that was selected to bind a modified thiazole orange
derivative with high affinity (3.4 nM) (Dolgosheina, et al. ACS
Chem Biol. 2014;9: 2412-2420).
[0165] RNA Mango used in conjunction with thiazol orange
derivatives have been used as both affinity purification tools, and
tools for visualization of RNA (Dolgosheina, et al. 2014). RNA
mango is commonly used within the context of a larger RNA scaffold,
and in particular the 6S RNA from bacteria.
[0166] In this example, RNA mango in the context of 6S RNA was
synthesized from 2 ss DNA oligonucleotides in one-step DNA template
assembly and transcription reactions. The first and second ss DNA
oligonucleotides had 15 nt regions at their 3' termini that were
complementary to each other. A first ss DNA oligonucleotide, 48 nt
in length, comprising the top strand of the T7 RNAP promoter and
the partial coding strand of the 6S RNA mango construct were
combined with a second ss DNA oligonucleotide, 200 nt in length,
made up of the template strand of the remainder of 6S mango under
conditions detailed above (FIG. 7A).
[0167] Reactions were carried out by combining the
oligonucleotides, enzymes, and buffers as described above and
incubating the reaction mixture at 37.degree. C. for 30 minutes.
Template assembly occurred via annealing of the 2 oligonucleotides
and their extension by an E. coli DNA polymerase I Klenow fragment
(FIG. 7B). Transcription began as ds DNA was formed in the reaction
(FIG. 7C). The 211 nt RNA products of the reaction were purified,
analyzed by gel electrophoresis (FIG. 8A-8D).
[0168] To demonstrate the function of the 6S RNA mango aptamer,
differing concentrations of 6S RNA mango were dispensed into tubes
in an 8 well strip in a buffer containing 10 mM Tris HCl pH 7.5,
160 mM KCl, and 1 micromolar TO1. After mixing, the tubes were
photographed on a UV transilluminator with a 312 nm light source
(FIG. 7D). Functional 6S Mango RNA was indicated by fluorescence
and was evident in the tubes containing RNA products from the
template assembly and transcription reaction.
[0169] It was concluded that the 211 nt configuration of RNA mango
was functional in conjunction with the TO1 thiazole orange
compound. RNA mango induced the fluorescence of TO1 upon binding.
The results suggest that RNA mango made in this way will be useful
for detection and visualization of RNA.
Example 7: Synthesis of functional RNA broccoli aptamers using DNA
template assembly and transcription in single reactions.
[0170] RNA broccoli is an aptamer that binds to and activates the
small molecule fluorophore DHBF1. RNA broccoli-DHBF1 complexes are
mimics of green fluorescent protein that can be used for RNA
tagging and detection, and localization by imaging (You, et al.
2015, Annu Rev Biophys. 2015; 44: 187-206).
[0171] An RNA containing 2 copies of diBroccoli in a scaffold of
F30 RNA (Filonov et al 2015 2015, Chem Biol. 2015;22: 649-660) was
synthesized as described above. The first and second ss DNA
oligonucleotides had 15 nt regions at their 3'-termini that were
complementary to each other. A first ss DNA oligonucleotide, 72 nt
in length, made up of the top strand of the T7 RNAP promoter and
the partial coding strand of the F30 diBroccoli RNA was combined
with a second ss DNA oligonucleotide, 200 nt in length, made up of
the template strand of the remainder of F30 diBroccoli RNA under
conditions detailed above and FIG. 8A.
[0172] Reactions were carried out by combining the
oligonucleotides, enzymes, and buffers as described in Example 1
and incubated at 37.degree. C. for 30 minutes. Template assembly
occurred via annealing of the 2 oligonucleotides and their
extension by E. coli DNA polymerase I Klenow fragment (FIG. 8B).
Transcription began as ds DNA was formed in the reaction (FIG. 8C).
The 235 nt RNA products of the reaction were purified and analyzed
by gel electrophoresis (FIG. 9).
[0173] To demonstrate the function of the F30 diBroccoli RNA,
differing concentrations of the reaction products were dispensed
into tubes in an 8 well strip in a buffer containing 10 mM Tris HCl
pH 7.5, 160 mM KCl, and 1 micromolar DHBF1. After mixing, the tubes
were scanned on a Typhoon multiimager with excitation at 457 nm and
emission detected at 526 nm (FIG. 8D). Functional F30 diBroccoli is
indicated by fluorescence and is evident in the tubes containing
RNA products from the template assembly and transcription reaction.
In this case the image is inverted so that stronger fluorescence
appears as a darker shade.
[0174] These examples are significant because they demonstrate the
usefulness of the method for the rapid generation of functional
aptamer RNAs from readily available and inexpensive ss DNA
oligonucleotide starting material. We envision that in addition, to
RNA aptamer production, that this method is useful for generating
pools of aptamers for selection, and for rapid prototyping and
testing of aptamer variants including minimers, functional modules,
scaffold variants, and aptamers that contain modified
nucleotides.
Example 8: Synthesis of a Pooled S. pyogenes Cas9 sgRNA Library
Using DNA Template Assembly and Transcription in a Single
Reaction.
[0175] In this example the method is used to a produce a pooled
library of S. pyogenes Cas9 sgRNAs. A pool of 54 nt first ss DNA
oligonucleotides can be synthesized that each contain a DNA
sequence module that corresponds to top strand of the T7 RNAP
promoter site, a sequence module that corresponds to a 20 nt-22 nt
targeting sequence for sgRNA being suitable for recognizing a
duplex DNA target site and a region of 14 nt complementary to the
repeat region of the S. pyogenes CRISPR repeat. In this example, 2
or more (up to 4.sup.22) ss DNA oligonucleotides are synthesized
each containing a different 20 nt-22 nt targeting region (although
there is in fact no upper limit on the number that might be
synthesized as needed). The library of first ss DNA oligonucleotide
can then be added to a reaction mixture containing a second ss DNA
oligonucleotide that contains the sequence module for the template
strand of the S. pyogenes tracrRNA linked to the remainder of the
S. pyogenes CRISPR repeat by a GAAA tetraloop sequence. First ss
DNA oligonucleotides can be commercially obtained from synthetic
oligonucleotide service providers (e.g. IDT) as pooled ss DNA
oligonucleotides, or alternatively can be obtained separately and
then pooled before combining with a second ss DNA oligonucleotide
as described in the method.
[0176] Reactions are carried out by combining the oligonucleotides,
enzymes, and buffers as described in Example 1 and incubating at
37.degree. C. for 30 minutes. Template assembly occurs via
annealing of one of the first oligonucleotides variants with one
copy of the second oligonucleotides and their extension by E. coli
DNA polymerase I Klenow fragment. Transcription begins as ds DNA is
formed in the reaction (FIG. 2A-2C).
[0177] This example demonstrates the utility of rapidly and
inexpensively creating pooled libraries of sgRNAs for use in
screening. Such libraries may include as few as 2 members, and as
many as practical restraints would allow (i.e. the scale of ss DNA
oligonucleotide input and size of template assembly and
transcription reaction required for complete representation in
sgRNA libraries of templates with extensive diversity). This
example also could be extended for use in screening a pool of
sequences encoding any functional RNA module. For example, RNA
aptamer variants, RNA stability elements, RNA targeting elements,
target sites for protein binding, and so on.
Sequence CWU 1
1
1129DNAArtificial SequenceSynthetic constructmisc_feature(7)..(23)n
is a, c, g, or t 1ttgacannnn nnnnnnnnnn nnntataat 29
* * * * *